Innovative Insights into In Vitro Activity of Colloidal Platinum Nanoparticles against ESBL-Producing Strains of Escherichia coli and Klebsiella pneumoniae

Abstract

Growing morbidity and mortality rates due to increase in the number of infections caused by MDR (multi-drug resistant) microorganisms are becoming some of the foremost global health issues. Thus, the need to search for and find novel approaches to fight AMR (antimicrobial resistance) has become obligatory. This study aimed to determine the antimicrobial properties of commercially purchased colloidal platinum nanoparticles by examining the existence and potency of their antibacterial effects and investigating the mechanisms by means of which they express these activities. Antimicrobial properties were investigated with respect to standard laboratory ATCC (American Type Cell Culture) and clinical extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia (E.) coli and Klebsiella (K.) pneumoniae. Standard microbiological methods of serial microdilution, modulation of microbial cell death kinetics ("time-kill" assays), and biofilm inhibition were used. Bacterial cell wall damage and ROS (reactive oxygen species) levels were assessed in order to explore the mechanisms of platinum nanoparticles' antibacterial activities. Platinum nanoparticles showed strong antibacterial effects against all tested bacterial strains, though their antibacterial effects were found to succumb to time kinetics. Antibiofilm activity was modest overall and significantly effective only against E. coli strains. By measuring extracellular DNA/RNA and protein concentrations, induced bacterial cell wall damage could be assumed. The determination of ROS levels induced by platinum nanoparticles revealed their possible implication in antibacterial activity. We conclude that platinum nanoparticles exhibit potent antibacterial effects against standard laboratory and resistant strains of E. coli and K. pneumoniae. Both, cell wall damage and ROS induction could have important role as mechanisms of antibacterial activity, and, require further investigation.

Keywords: antibacterial agents; antimicrobial nanoparticles; bacterial resistance; multidrug-resistant bacteria; nanomedicine; nanopharmaceuticals; platinum nanoparticles.

Figure 1

Number of colonies of Klebsiella pneumoniae ESBL+ MFBF 10690 over time (h) treated with nPt (c = 2 × MIC) relative to the control. CFU—colony forming unit; t—time.

Figure 2

Effect of various concentrations of platinum nanoparticles on biofilm formation of (a) E. coli ATCC and E. coli ESBL and (b) K. pneumoniae ATCC and K. pneumoniae ESBL compared to the control. A_570nm—absorbance at 570 nm wavelength; PC—positive control (untreated bacteria); NC—negative control (sterile medium). * statistically significant difference (p < 0.05)

Figure 3

(a) Effects of various concentrations of platinum nanoparticles on preformed (mature) biofilms of E. coli ATCC and E. coli ESBL compared to the control. (b) Effects of various concentrations of platinum nanoparticles on preformed (mature) biofilms of K. pneumoniae ATCC and K. pneumoniae ESBL compared to the control. A_570nm—absorbance at 570 nm wavelength; PC—positive control (untreated bacteria); NC—negative control (sterile medium).

Figure 4

Comparison of bacterial planktonic cell survivability after 48 and 72 h of platinum nanoparticle exposure. Values are expressed as means ± standard deviations. c—concentration.

Figure 5

Interrelations of ROS concentrations in the extracellular compartments of all samples and time dependence for all tested bacterial strains. nanoPt*—platinum nanoparticles.

Figure 6

Interrelations of extracellular DNA/RNA concentrations over time for all tested bacterial strains. nanoPt*—platinum nanoparticles.

Figure 7

Graphical representation of DNA/RNA leakage (release) dynamics for E. coli ATCC 10536 at several time points of measurement over a 24 h period. The average values measured for each time point for samples of bacteria treated with 2 × MIC of nPt, bacteria treated with ethanol (as positive control), and untreated bacteria (inoculum, i.e., the negative control) are shown (along with standard deviations) and compared.

Figure 8

Interrelation of extracellular protein concentrations over time for all tested bacterial strains. nanoPt*—platinum nanoparticles.

Figure 8

Interrelation of extracellular protein concentrations over time for all tested bacterial strains. nanoPt*—platinum nanoparticles.

Figure 9

Nanoparticle Tracking Analyzer data obtained after analysis of original colloidal platinum nanoparticle samples: (a) distribution of particle sizes by concentration; (b) distribution of particle sizes by intensity (n = 3).